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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from authoritative database.

Data source

Reference
Reference Type:
other: authoritative database
Title:
Genetic toxicity study for the given test chemical by using Salmonella typhimurium strain.
Author:
National Institute of Technology and Evaluation
Year:
2010
Bibliographic source:
Japan chemicals collaborative knowledge database (J-check)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
as per Prival modification (MJ Prival, and VD Mitchell, 1982)
Principles of method if other than guideline:
To evaluate the mutagenic potential of test chemical in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): 2-Naphthalenecarboxylic acid, 3-hydroxy-4-[(4-methyl-2-sulfophenyl)azo]-, calcium salt
- Molecular formula (if other than submission substance): C18H14N2O6S.Ca
- Molecular weight (if other than submission substance): 424.445 g/mole
- Smiles notation (if other than submission substance): c12c(c(c(C(=O)[O-])cc1cccc2)O)\N=N\c1c(cc(C)cc1)S(=O)(=O)[O-].[Ca+2]
- InChl (if other than submission substance): 1S/C18H14N2O6S.Ca/c1-10-6-7-14(15(8-10)27(24,25)26)19-20-16-12-5-3-2-4-11(12)9-13(17(16)21)18(22)23;/h2-9,21H,1H3, (H,22,23)(H,24,25,26);/q;+2/p-2/b20-19+;
- Substance type: Organic
- Physical state: Solid
purity: 99%,

Method

Target gene:
Histidine for Salmonella typhimurium and tryptophan Escherichia coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: NADH 4 μmol, Magnesium chloride 8 μmol, NADPH 4 μmol, Potassium chloride 33 μmol, 0.2 M phosphate buffer (pH 7.4) 1000 μmol, Glucose · 6-phosphate 5 μmol, FMN 2 μmol
- source of S9 : Kikkoman Corporation
- method of preparation of S9 mix : S9 was prepared from non-induced liver of 8-week-old Syrian Hamster (Std: Syrian) males used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.3 ml
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test chemical was stable in DMSO solution, and the content of test substance in preparation liquid was within a predetermined value range.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: -S9, AF-2 (TA100, WP2, TA98), +S9, 2-aminoanthracene (all strains), trypane blue (TA100, TA98, azo reduction method)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium (Standard method of Ames and Azo reduction method)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: The pre-incubation method was used to test by direct method and metabolic activation method.
- Exposure duration/duration of treatment: 48 hours (Standard method of Ames); 20 min (Azo reduction method)
- Harvest time after the end of treatment (sampling/recovery times): No data
Evaluation criteria:
Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control , and when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system.
Statistics:
Yes, Mean ±standard deviation was observed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: S. typhimurium TA100, TA1535, TA98, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable): When test chemical was tested at a common ratio of approximately 3 in the range of 50 to 5000 μg / plate, no antibacterial activity was observed in either direct method or metabolic activation method of all test bacteria. From the above results, it was decided that the maximum dose in this test was set to 5000 μg / plate in all the test bacteria, in the direct method and the metabolic activation method, and the dose was set to 5 in the common ratio 2.
Remarks on result:
other: No mutagenic effects were observed.

Any other information on results incl. tables

Please refer attached background material

Applicant's summary and conclusion

Conclusions:
Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. The test result was considered to be negative in all strain in the presence and absence of metabolic activation S9.
Executive summary:

Genetic toxicity in vitro study was assessed for the given test chemical according to Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and as per Prival modification (MJ Prival, and VD Mitchell, 1982). The test chemical was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9 (0.3 ml) prepared from non-induced liver of 8-week-old Syrian Hamster (Std: Syrian) males was used with composition of NADH 4 μmol, Magnesium chloride 8 μmol, NADPH 4 μmol, Potassium chloride 33 μmol, 0.2 M phosphate buffer (pH 7.4) 1000 μmol, Glucose · 6-phosphate 5 μmol, FMN 2 μmol at test concentrations of 0, 312.5, 625, 1250, 2500, 5000 µg/plate. Test chemical was dissolved in DMSO. The positive control substances used are as follows, AF-2 : Frill Furide, SA : Sodium azide, 9-AA : 9-aminoacridine, 2-AA : 2-aminoanthracene and TB : Trypan blue. The study was conducted by Standard method of Ames and Azo reduction method. In Azo reduction method, the pre-incubation method was used to test by direct method and metabolic activation method. 0.1 ml of the test bacterial solution, 0.1 ml of the test substance preparation solution, 0.5 ml of the phosphate buffer solution (0.5 ml of the S9 mixed solution in the metabolic activation method)) were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes After it was done, 2 ml of top agar was added and mixed, and it was poured onto a synthetic medium flat plate. At the same time, a control test was conducted using a solvent or two kinds of positive control substance solutions instead of the test substance preparation solution. When test chemical was tested at a common ratio of approximately 3 in the range of 50 to 5000 μg / plate, no antibacterial activity was observed in either direct method or metabolic activation method of all test bacteria. From the above results, it was decided that the maximum dose in this test was set to 5000 μg / plate in all the test bacteria, in the direct method and the metabolic activation method, and the dose was set to 5 in the common ratio 2. Among the five test bacteria used, in the direct method or metabolic activation method of one or more test bacteria, the number of revertive mutant colonies on the flat plate containing the test substance is more than twice that of the negative control, and when the increase was found to be reproducible or dose-dependent, it was decided that the test substance had mutagenicity (positive) in this test system. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA by Ames test. Hence the substance cannot be classified as gene mutant in vitro.